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Molecular Challenges in the Diagnosis of X-Linked Chronic Granulomatous Disease: CNVs, Intronic Variants, Skewed X-Chromosome Inactivation, and Gonosomal Mosaicism

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Abstract

Chronic granulomatous disease (CGD) is a prototypical inborn error of immunity affecting phagocytes, in which these cells are unable to produce reactive oxygen species. CGD is caused by defects in genes encoding subunits of the NADPH oxidase enzyme complex (CYBA, CYBB, CYBC1, NCF1, NCF2, NCF4); inflammatory responses are dysregulated, and patients are highly susceptible to recurrent severe bacterial and fungal infections. X-linked CGD (XL-CGD), caused by mutations in the CYBB gene, is the most common and severe form of CGD. In this study, we describe the analytical processes undertaken in 3 families affected with XL-CGD to illustrate several molecular challenges in the genetic diagnosis of this condition: in family 1, a girl with a heterozygous deletion of CYBB exon 13 and skewed X-chromosome inactivation (XCI); in family 2, a boy with a hemizygous deletion of CYBB exon 7, defining its consequences at the mRNA level; and in family 3, 2 boys with the same novel intronic variant in CYBB (c.1151 + 6 T > A). The variant affected the splicing process, although a small fraction of wild-type mRNA was produced. Their mother was a heterozygous carrier, while their maternal grandmother was a carrier in form of gonosomal mosaicism. In summary, using a variety of techniques, including an NGS-based targeted gene panel and deep amplicon sequencing, copy number variation calling strategies, microarray-based comparative genomic hybridization, and cDNA analysis to define splicing defects and skewed XCI, we show how to face and solve some uncommon genetic mechanisms in the diagnosis of XL-CGD.

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Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We are deeply grateful to the affected individuals who participated in this study and their families. We acknowledge Celine Cavallo for extensive revision of language and grammar. This research is supported by the European Reference Network for Rare Immunodeficiency, Autoinflammatory and Autoimmune Diseases (ERN-RITA).

Funding

This study was funded by Instituto de Salud Carlos III, grants PI18/00346 and PI20/00761, co-financed by the European Regional Development Fund (ERDF), and by the Jeffrey Modell Foundation. Laura Batlle-Masó has received research support from European Union-NextGenerationEU, Ministry of Universities and Recovery, Transformation and Resilience Plan, through a call from Pompeu Fabra University (Barcelona).

Author information

Authors and Affiliations

  1. Infection and Immunity Research Group, Vall d’Hebron Research Institute (VHIR), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Catalonia, Spain

    Laura Batlle-Masó, Jacques G. Rivière, Andrea Martín-Nalda, Marina Garcia-Prat, Alba Parra-Martínez & Pere Soler-Palacín

  2. Pediatric Infectious Diseases and Immunodeficiencies Unit, Vall d’Hebron Children’s Hospital (HUVH), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Catalonia, Spain

    Laura Batlle-Masó, Jacques G. Rivière, Andrea Martín-Nalda, Marina Garcia-Prat, Alba Parra-Martínez & Pere Soler-Palacín

  3. Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Barcelona, Catalonia, Spain

    Laura Batlle-Masó, Jacques G. Rivière, Clara Franco-Jarava, Andrea Martín-Nalda, Marina Garcia-Prat, Alba Parra-Martínez, Aina Aguiló-Cucurull, Mónica Martinez-Gallo, Pere Soler-Palacín & Roger Colobran

  4. Pompeu Fabra University (UPF), Barcelona, Catalonia, Spain

    Laura Batlle-Masó

  5. Translational Immunology Research Group, Vall d’Hebron Research Institute (VHIR), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Catalonia, Spain

    Clara Franco-Jarava, Aina Aguiló-Cucurull, Mónica Martinez-Gallo & Roger Colobran

  6. Immunology Division, Vall d’Hebron University Hospital (HUVH), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Catalonia, Spain

    Clara Franco-Jarava, Aina Aguiló-Cucurull, Mónica Martinez-Gallo & Roger Colobran

  7. Department of Clinical and Molecular Genetics, Vall d’Hebron University Hospital (HUVH), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Catalonia, Spain

    Neus Castells & Roger Colobran

  8. Medicine Genetics Research Group, Vall d’Hebron Research Institute (VHIR), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Catalonia, Spain

    Neus Castells

  9. Department of Cell Biology, Physiology and Immunology, Autonomous University of Barcelona (UAB), Bellaterra, Catalonia, Spain

    Roger Colobran

Authors
  1. Laura Batlle-Masó
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  2. Jacques G. Rivière
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  3. Clara Franco-Jarava
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Contributions

LBM, PSP, and RC were responsible for the study design. JGR, AMN, and PSP provided patient care, and collected and provided clinical data. AAC performed sample collection and Sanger sequencing. MMG was responsible for flow cytometry studies. CFJ, MGP, and APM performed laboratory analyses and molecular studies. NC was responsible for microarray-based comparative genomic hybridization data. LBM and RC analyzed and interpreted the data, and wrote the manuscript. PSP and RC designed and supervised the project, provided resources, and edited the manuscript. All co-authors reviewed, commented on, and approved the final version of the manuscript.

Corresponding authors

Correspondence to Pere Soler-Palacín or Roger Colobran.

Ethics declarations

Ethics Approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Vall d’Hebron University Hospital [date: 11/06/2021 / code: PR(AG)202/2021].

Consent to Participate

Informed consent was obtained from all individual participants included in the study. For pediatric patients (under 18 years old), written informed consent was obtained from the parents.

Consent for Publication

The authors affirm that human research participants provided informed consent for publication of data contained in this manuscript.

Conflict of Interest

The authors declare no competing interests.

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Pere Soler-Palacín and Roger Colobran are senior corresponding authors.

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Batlle-Masó, L., Rivière, J.G., Franco-Jarava, C. et al. Molecular Challenges in the Diagnosis of X-Linked Chronic Granulomatous Disease: CNVs, Intronic Variants, Skewed X-Chromosome Inactivation, and Gonosomal Mosaicism. J Clin Immunol 43, 1953–1963 (2023). https://doi.org/10.1007/s10875-023-01556-x

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